This research is examines mechanisms by which membranes, particularly matrix vesicles, promote biological mineralization. Various components of the membrane, including ion-transporting proteolipids, phospholipids, alkaline phosphatase and phospholipase A2, are involved in perturbation of the system by vitamin D provides an excellent opportunity to dissect the membrane-dependent mineralization process. The research proposed will use a chondrocyte culture model to address the following Specific Aims. (1) To determine whether chondrocytes produce vitamin D metabolites as paracrine or autocrine regulators of cell and matrix vesicle function. Regulation of 14C-25-(OH)D3 metabolism by exogenous 1,25-(OH)2D3, 24,25-(OH)2D3, TGFb and dexamethasone will be characterized. The presence of radiolabeled metabolite in the plasma isolated membranes will indicate whether secreted metabolites can act as autocrine or paracrine agents. (2) To examine effects of vitamin D metabolites on membrane signaling systems. Effects of Vitamin D metabolites on inositol phosphate turnover, acylation and reacylation of phospholipids, production of endogenous PGE2 ( and membrane response to exogenous PGE2), and membrane fluidity will be studied. (3) To characterize the membrane effects of vitamin D metabolites on functional properties of matrix vesicles. Steroid hormone effects on kinetic properties of purified phosphatidyinositol-linked and phosphatidyl inositol-free alkaline phosphatase activity will be compared to that in native matrix vesicles. These results will be correlated with effects of steroid hormones on matrix vesicle mineralization in culture and in vitro. The chondrocyte culture model affords an opportunity to better understand mechanisms of vitamin D action in chondrogenesis and cartilage cell metabolism. The studies have immediate implications in fracture healing, osteoporosis, vitamin D deficiency diseases and normal bone growth and development.